The proliferation and differentiation of cells in multicellular organisms is subject to a highly regulated process. A distinguishing feature of cancer cells is the absence of control over this process; proliferation and differentiation become deregulated resulting in uncontrolled growth. Significant research efforts have been directed toward better understanding this difference between normal and tumor cells. One area of research focus is growth factors and, more specifically, autocrine growth stimulation.
Growth factors are polypeptides which carry messages to cells concerning growth, differentiation, migration and gene expression. Typically, growth factors are produced in one cell and act on another cell to stimulate proliferation. However, certain malignant cells, in culture, demonstrate a greater or absolute reliance on an autocrine growth mechanism. Malignant cells which observe this autocrine behavior circumvent the regulation of growth factor production by other cells and are therefore unregulated in their growth.
Study of autocrine growth control advances understanding of cell growth mechanisms and leads to important advances in the diagnosis and treatment of cancer. Toward this end, a number of growth factors have been studied, including insulin-like growth factors (“IGF1” and “IGF2”), gastrin-releasing peptide (“GRP”), transforming growth factors alpha and beta (“TGF-a” and (“TGF”), and epidermal growth factor (“EGF”).
The present invention is directed to a recently discovered growth factor. This growth factor was first discovered in the culture medium of highly tumorigenic “PC cells,” an insulin-independent variant isolated from the teratoma derived adipogenic cell line 1246. This growth factor is referred to herein as “GP88.” GP88 has been purified and structurally characterized. Amino acid sequencing of GP88 indicates that GP88 has amino acid sequence similarities with the mouse granulin/epithelin precursor.
Granulins/epithelins (“grn/epi”) are 6 kDa polypeptides and belong to a novel family of double cysteine rich polypeptides. U.S. Pat. No. 5,416,192 (Shoyab et al.) is directed to 6 kDa epithelins, particularly epithelin 1 and epithelin 2. According to Shoyab, both epithelins are encoded by a common 63.5 kDa precursor, which is processed into smaller forms as soon as it is synthesized, so that the only natural products found in biological samples are the 6 kDa forms. Shoyab et al. teaches that the epithelin precursor is biologically inactive.
Contrary to the teachings of Shoyab et al., the present inventor's laboratory has demonstrated that the precursor is not always processed as soon as it is synthesized. Studies, conducted in part by this inventor, have demonstrated that the precursor (i.e., GP88) is in fact secreted as an 88 kDa glycoprotein with an N-linked carbohydrate moiety of 20 kDa. Analysis of the N-terminal sequence of GP88 indicates that GP88 starts at amino acid 17 of the grn/epi precursor, demonstrating that the first 17 amino acids from the protein sequence deduced from the precursor cDNA correspond to a signal peptide compatible with targeting for membrane localization or for secretion. In contrast to the teachings of Shoyab et al., GP88 is biologically active and has growth promoting activity, particularly as an autocrine growth factor for the producer cells.
Breast cancer is a major worldwide cause of morbidity and mortality among women. Estrogen is known to be a primary stimulator for estrogen receptor positive (ER+) human breast cancer cell growth in vivo and in vitro. Although estrogen is initially required for establishment and proliferation of breast tumors, the development of estrogen-independent tumors during the course of breast cancer is indicative of poor prognosis. It has been postulated that the mitogenic effect of estrogen in breast cancer cells is mediated, at least partially, by autocrine growth factors, including growth factors regulated by estrogen. Thus, the identification and characterization of estrogen-responsive genes, particularly genes encoding growth factors, contributes to the understanding of the effects of estrogen in breast cancer cells.
Tamoxifen citrate (“tamoxifen”) is a nonsteroidal antiestrogen commonly prescribed to patients suffering from breast cancer that has demonstrated potent antiestrogenic and antineoplastic properties. See U.S. Pat. No. 4,536,516. Tamoxifen is an estrogen receptor antagonist that competes with estrogen for binding to estrogen receptors. Other antiestrogens include, raloxifene, aromatase inhibitors (e.g., Arimidex® (anastrozole), Femera®), and estrogen receptor down-regulators (e.g., Faslodex®). The antiestrogenic effects of tamoxifen may be related to its ability to compete with estrogen for binding sites in target tissues. Other antiestrogens, such as aromatase inhibitors, inhibit or reduce the amount of estrogen available. For example, aromatase inhibitors prevent the conversion of androgen into estrogen thereby reducing the amount of estrogen available. Estrogen receptor down regulators inhibit or reduce the number of estrogen receptors on the cell.
Tamoxifen is currently available in 10 and 20 mg tablets from AstraZeneca and Barr Laboratories under the brand name Nolvadex® and is indicated for metastatic breast cancer, adjuvant therapy of breast cancer, ductal carcinoma in situ, and reduction of breast cancer incidence in women at high risk for breast cancer. Thus, tamoxifen can be used to treat and prevent various cancers and particularly breast cancer.
Tamoxifen administration, however, carries with it potentially serious risks for the patient. For example, tamoxifen administration has been associated with an increased risk of ovarian cancer. Moreover, some patients exhibit resistance to the intended beneficial effects of tamoxifen. Therefore, administration of tamoxifen to a patient who is resistant to its benefits causes unnecessary harm to the patient by increasing the risk of ovarian cancer and at the same time delaying or causing the patient to forego more effective treatments.
Accordingly, tamoxifen administration should be limited to those patients who are most likely to benefit from treatment with tamoxifen. An accurate determination of whether a patient will be susceptible or resistant to the antineoplastic effects of tamoxifen administration, before embarking on such a treatment course, would be a valuable diagnostic tool.
The art currently teaches that the absence of estrogen receptor in a patient corresponds with tamoxifen resistance. Cancer patients are routinely tested for the presence or absence of the estrogen receptor in an attempt to predict whether the patient's will be resistant or responsive to tamoxifen therapy. Based on the current test, cancer patients who tests positive for the presence of estrogen receptors (“ER+” or “estrogen receptor positive patients”) are typically prescribed tamoxifen.
However, a significant number of ER+ patients are in fact resistant to tamoxifen. Thus, routinely prescribing tamoxifen for ER+ patients may be harmful, and increase the patient's risk of developing ovarian or other forms of cancer.